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Stability-indicating HPTLC method for simultaneous determination of Ketoprofen, Methyl Paraben and Propyl Paraben in gel formulation
j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 9 4 5 e9 5 3
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Stability-indicating HPTLC method for simultaneous determination of Ketoprofen, Methyl Paraben and Propyl Paraben in gel formulation Pallavi Mangesh Patil a,*, Sagar Baliram Wankhede b, Praveen Digambar Chaudhari c a
Centre for Research and Development, Prist University, Vallam, Thanjavur, Tamil Nadu 613403, India Department of Pharmaceutical Chemistry, Padm. Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune 411018, Maharashtra, India c Department of Pharmaceutics, P.E. Society’s Modern College of Pharmacy, Yamunanagar, Nigdi, Pune 411044, Maharashtra, India b
article info
abstract
Article history:
Aim: A novel and quick HPTLC-densitometric method was developed for the simultaneous
Received 20 August 2013
determination of Ketoprofen, Methyl Paraben, and Propyl Paraben.
Accepted 2 September 2013
Methods: Chromatographic separation of the drugs was performed on precoated silica gel 60
Available online 26 September 2013
F254 Merck plates using Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) as a mobile phase. A TLC scanner set at 265 nm was used of Ketoprofen, Methyl Paraben, Propyl
Keywords:
Paraben respectively were validated according to ICH guidelines. Forced degradation con-
Ketoprofen (KETO)
ditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress,
Methyl Paraben (MP)
as suggested in the ICH guideline Q1A (R2).
Propyl Paraben (PP)
Results: The three drugs were satisfactorily resolved with Rf values of 0.33 0.05,
HPTLC
0.54 0.05, 0.71 0.05 for Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Cali-
Force degradation studies
bration curves were polynomial in the range 200e1000 ng/band, 200e1500 ng/band, 100 e600 ng/band, for Ketoprofen, Methyl Paraben, and Propyl Paraben respectively. Correlation coefficient (r) values were 0.9917, 0.9927, 0.9906 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. The percentage recovery ranges from 99 to 101%. Conclusion: A low relative standard deviation (<2%) was found for both precision and robustness study showing that the proposed method was precise and robust. The method had an accuracy of 99.95%, 99.85% and 100.07 of Ketoprofen, Methyl Paraben, Propyl Paraben respectively were validated according to ICH guidelines. The drug showed instability in oxide , heat and UV light, while it remained stable in neutral conditions. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. Tel.: þ91 9823720695 (mobile). E-mail addresses: [email protected], [email protected] (P.M. Patil). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.09.004
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1.
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Introduction
A new pharmaceutical preparation (gel) containing ketoprofen (Fig .1) as an active compound with anti-inflammatory and analgesic activity was developed for treatment of diseases of the muscolo-skeletal apparatus, in which a local action is preferred. In order to prevent bacterial growth during the storage of the formulation,1,2 two commonly used preservativesda mixture of the methyl ester and propyl ester of phydroxybenzoic acid Methyl Paraben (MP) (Fig. 2) and Propyl Paraben (PP) (Fig. 3)dhave been used gas chromatographyemass spectrometry (GCeMS),3 capillary electro chromatography,4,5 high-performance liquid chromatography (HPLC) 6e8 , HPLCeMS9,10 or micellar chromatography11 as well. Only one HPLC method has been found in literature12 for simultaneous determination of KP and its degradation products, but not in the presence of preservatives. Recently, preservatives in pharmaceuticals have to be quantified. HPLC analysis of MP and PP is frequently described in the literature13e15; another publication deals with simultaneous quantification of Ketoprofen and Parabens in a commercial gel formulation by RPeHPLC with UV detection,16 but there is no any HPTLC method describing simultaneous determination of all three componentsdketoprofen, MP and PPdin pharmaceutical preparations with no any HPTLC method describing simultaneous determination in this mobile phase with beneficial system suitability parameter. For such a formulation, a novel method capable to analyze simultaneously the active component ketoprofen, and its two preservatives Methyl Paraben and Propyl Paraben was developed. Thereafter, this HPTLC method17 was successfully applied for the separation, quantification and stability study of all these compounds in formulated ketoprofen gel 2.5%.
Fig. 1 e Ketoprofen.
Fig. 2 e Methyl Paraben.
2.2.
Preparation of standard stock solutions
2.2.1.
Stock solution A
Accurately weighed quantity (100 mg) of KETO was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.2.2.
Stock solution B
2.
Material and method
Accurately weighed quantity (100 mg) of MP was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.1.
Reagents and chemicals
2.2.3.
Toluene, Ethyl acetate, Glacial acetic acid from S. D. Fine Chemicals, Mumbai Reference standard Ketoprofen and Methyl Paraben and Propyl Paraben were procured from ZIM laboratories, Nagpur, India as gift samples. Formulated gel formulation (Ketoprofen 2.5% w/w). Instrumentation and chromatographic conditions are given in the following table:
Sr. no.
Instruments
Descriptions
1 2 3 4 5
HPTLC system Sample application Scanner Software Saturated chamber
6
HPTLC plate
7
Syringe
Camag HPTLC system Camag Linomat IV automatic sample Camag TLC scanner Camag winCATS software Camag twin-trough chamber (10 10) and (20 20) Merck HPTLC plate coated with silica gel 60 F 254 (0.2 mm thickness) on aluminum sheet Hamilton syringe (100 ml)
Stock solution C
Accurately weighed quantity (100 mg) of PP was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.2.4.
Stock solution D
An accurately weighed quantity of 250 mg KETO and 100 mg MP, 10 mg was transferred to 100.0 mL volumetric flasks, 40.0 mL of mobile phase was added; the content was dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 10.0 mL volumetric flask and diluted to the mark with mobile phase. Further, 5.0 mL of
Fig. 3 e Propyl Paraben.
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above solution was diluted to 10.0 mL with mobile phase (concentration of 625 mg/mL KETO and 250 mg/mL MP, 25 mg/mL PP respectively). The solution was mixed and filtered through 0.2 m membrane filter.
2.3.
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Migration distance: z80 mm Temperature: 20 5 C Scanning mode: Absorbance/Reflectance Slit dimensions: 5 0.45 mm Scanning wavelength: 265 nm
Selection of mobile phase The retention factors of KETO, MP and PP were:
Aliquot portion of standard stock solutions D (5 mL each) was applied on TLC plates in the form of band (band size: 6 mm). Different solvents with varying polarity as well as combination of solvent were tried to get well separated bands of the drugs. After trying several permutations and combinations, the solvent system containing Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) was found to be most satisfactory as it gave good resolution of both drugs.
2.4. Selection of wavelength for densitometric evaluation of separated bands Standard stock solution D was applied on TLC plate with the help of CAMAG LINOMAT-V automatic sample applicator, the plate was chromatographed in twin-through glass chamber saturated with mobile phase for 30 min. After chromatographic development, the plate was removed and air dried. The separated bands on the TLC plate were scanned over the wavelength range of 200e700 nm. The wavelength 265 nm was selected for densitometric evaluation of separated bands. The overlain spectrum obtained is depicted in Fig. 4.
2.5.
Optimum chromatographic conditions
Stationary phase: Aluminum plates precoated with silica gel 60 F254 (Merck) Mobile phase: Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) Plate size: 10 cm 10 cm Mode of application: Band Band size: 6 mm (Distance between two bands: 5.5 mm) Sample volume: 5 mL Development chamber: Twin-through glass chamber, 10 cm 10 cm with (20 20) stain less steel lid Saturation time: 30 min Se´paration technique: Ascending
KETO: 0.33 0.05 MP: 0.54 0.05 PP: 0.71 0.05 Densitogram of KETO, MP and PP is shown in Fig. 5.
2.6.
Study of linearity range
The standard stock solution A containing KETO and standard stock solution B containing MP and stock solution C containing PP was applied on the TLC plate in the range 1e6 mL with the help of micro syringe using LINOMAT-V automatic sample applicator. The plate was then developed and scanned under the above mentioned chromatographic conditions. Rf was recorded for each drug concentration and the calibration curves of the concentration vs. Rf were constructed for both the drugs. The calibration curve for KETO and MP and PP are depicted in Figs. 6e8 respectively.
2.6.1.
Selection of mobile phase
From standard stock D was appropriately to obtain final concentration 625 mg/mL KETO and 250 mg/mL MP, 25 mg/mL PP respectively. The diluted standard solutions were filtered through 0.2 m membrane filter. After trying several permutations and combinations, the solvent system containing Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) was found to be most satisfactory as it gave good resolution of both drugs.
2.7.
Preparation of gel formulation of Ketoprofen
Ketoprofen gel formulation was prepared using 1% carbopol 940 and as a gelling agent. Gelling agent was dispersed in a small quantity of distilled water 75 ml and then stored overnight to ensure complete hydration. Ketoprofen in a suitable
Fig. 4 e Overlain spectra of KETO, MP and PP.
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Fig. 5 e Typical densitogram of KETO, MP and PP.
solvent (water) as added to the dispersion and make up weight with distilled water. Other excipients (Methyl Paraben 1% and propyl Paraben 0.1%) were also added slowly with continuous stirring. In carbopol gels, pH of the vehicle was brought to neutral by using TEA (Triethanolamine). The final weight of the gel was adjusted to 100 gm with distilled water. Entrapped air bubbles were removed by keeping the gels in vacuum desiccators as shown in Table 1.
2.8.
Analysis of gel formulation
An accurately weighed quantity of gel was weighed equivalent to about 1000 mg of Ketoprofen and 400 mg of Methyl Paraben and 40 mg Propyl Paraben into a 1000 mL volumetric flask. And appropriate amount of methanol was then added. The mixture was ultrasonicated for 30 min with heating and allowed to cool at room temperature before adjusting to volume with methanol. The organic layer was decanted and the extraction procedure was repeated. The resulting mixture was centrifuged at 3500 rpm for 20 min and 20 mL of the clear supernatant was injected directly
Fig. 6 e Standard calibration curve for Ketoprofen.
onto the column. After centrifugation 20 mL of this mixture was injected into the chromatograph. The resulting solution was mixed and filtered through Whatman filter paper and filtrate was appropriately diluted to get approximate concentration and to obtain final concentration of 1000 mg/mL KETO and 400 mg/mL MP, 40 mg/mL respectively. The diluted solution was filtered through 0.20 m filter. On the TLC plate two bands of standard stock solution D and four bands of sample solution, 5.0 mL each, were applied and the plate was developed and scanned under the optimum chromatographic condition. After chromatographic development the peak obtained for standard and sample bands was integrated. The amount of KETO, MP and PP present in applied volume of standard solution was fed to computer. Amount of drug present in applied volume of sample solution was obtained by comparing Rf of sample bands with that of standard bands. Amount of drug estimated in mg/gel and the percent label claim were calculated using the following formula: The content of KETO, MP and PP in sample was calculated using the following formula no. 1.
Fig. 7 e Standard calibration curve for Methyl Paraben.
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2.9.1.
Accuracy
To as certain the accuracy of proposed method, recovery studies were carried out by standard addition method, as per ICH guidelines.
2.10.
Preparation of sample solutions
An accurately weighed quantity of pre-analyzed gel equivalent to about 1000 mg KETO, 400 mg MP and 40 mg PP was transferred individually in nine different 1000.0 mL volumetric flasks. To each of the flask following quantities of KETO, MP and PP were added: Fig. 8 e Standard calibration curve for Propyl Paraben.
Table 1 e Composition of the carbopol and pure drug Ketoprofen as below. Ingredient
Quantity taken
Ketoprofen Methyl Paraben Propyl Paraben Carbopol (1%) as gel base Double distilled water Triethanolamine
2.5 g 1.0 g 0.1 g QS Make up to 100 ml Q S to neutralize gel
Q.S (Quality sufficient).
Amount of drug estimated ðmg=gelÞ ¼
Mean amount estimated ðmgÞ in applied volume Volume of sample solution applied ðmLÞ
Volume of stock solution ðmLÞ Wt: of gel taken ðmgÞ
Average wt: of gel ðmgÞ
(1)
Percent label claim was calculated using above formula no 1. Results of analysis of gel formulation and its statistical evaluation are shown in Tables 2 and 3 respectively.
2.9.
Method validation
The proposed method was validated by studying several parameters such as accuracy, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and robustness.
Flask no.1: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.2: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.3: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.4: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.5: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.6: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.7: 1200 mg KETO þ 480 mg MP þ 48 mg PP Flask no.8: 1200 mg KETO þ 480 mg MP þ 48 mg PP Flask no.9: 1200 mg KETO þ 480 mg MP þ 48 mg PP Then 100 mL methanol was added to each flask and content of the flask was ultrasonicated for 20 min, volume was then made up to the mark with mobile phase. The solution was individually mixed and filtered through Whatman filter paper no. 42. From the filtrate, 1.0 mL solution was diluted to 10.0 mL with mobile phase. The diluted solution was filtered through 0.2 m membrane filter. On the TLC plate two bands of standard stock solution D and four bands of sample solution, 5.0 mL each, were applied and the plate was developed and scanned under the optimum chromatographic condition. After chromatographic development the peak obtained for standard and sample bands were integrated. The amount of KETO, MP and PP present in applied volume of standard solution was fed to computer. Amount of drug present in applied volume of sample solution was obtained by comparing Rf of sample bands with that of standard bands. Amount of KETO, MP and PP in sample was calculated by comparing the mean Rf for standard and sample solution by formula no. 2. Amount of KETO, MP and PP in sample (mg) was calculated by following formula:
Table 2 e Results of analysis gel formulation. Formulated gel Sr. no.
1. 2. 3. 4. 5. 6.
Average weight: 4.000 mg
Weight of gel taken (mg)
4000 4000 4000 4000 4000 4000
Amount of drug estimated (mg/Gel taken)
% Label claim
KETO
MP
PP
KETO
MP
PP
1000.09 1000.01 1000.00 999.98 999.78 1000.01
399.00 399.12 400.00 400.12 398.78 401.02
40.00 39.87 39.87 40.00 41.02 41.77
100.90 100.49 100.00 99.88 99.79 100.03
99.80 99.87 100.00 100.12 99.88 100.73
100.00 99.89 99.89 100.00 101.00 101.01
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Table 3 e Statistical validation for analysis of gel formulation. Sr. no.
Drug
Amount of drug estimated (mg/gel)a
% Label claima
S.D
C.V
S.E
1. 2. 3
KETO MP PP
999.99 399.67 40.42
99.95 99.85 100.00
1.3143 1.2108 0.2147
1.4278 1.2146 0.2251
0.3181 0.1504 0.1151
a Mean of six determinations.
Table 4 e Results of recovery studies. Level of recover
Weight of gel taken (mg)
80%
Amount of drug added (mg)
Amount of drug recovered (mg)
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
800 800 800 1000 1000 1000 1200 1200 1200
320 320 320 400 400 400 480 480 480
32 32 32 40 40 40 48 48 48
799.78 800.01 800.12 999.18 999.70 1000.10 1198.78 1200.00 1201.03
318.13 319.79 320.11 399.18 399.77 400.18 479.11 480.72 480.75
31.99 32.00 32.11 40.10 39.98 40.11 47.91 47.95 48.01
99.71 100.01 100.19 99.78 99.88 100.02 99.93 100.00 101.02
98.11 99.89 100.11 99.77 99.85 100.11 99.10 100.42 100.47
99.98 100.00 100.12 100.11 99.92 100.12 99.92 99.95 100.01
4000 4000 4000 4000 4000 4000 4000 4000 4000
100%
120%
% Recovery
Table 5 e Statistical validation for recovery study. % Mean recoverya
Level of recovery
80% 100% 120%
Standard deviation
% R.S.D.
S.E
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
99.97 99.95 100.02
99.89 99.85 100.15
100.10 100.05 100.15
1.2679 1.2454 1.2788
1.2488 1.2214 1.2958
1.3011 1.3117 1.2481
1.2519 1.2316 1.2668
1.2310 1.2161 1.2848
1.2923 1.3109 1.2316
0.6111 0.5922 0.7155
0.5977 0.5811 0.7066
0.7122 0.7211 0.6811
a Mean of three determinations.
Amount of drug KETO; MP and PP ðmLÞ estimated ðmgÞ
2.11.2. Inter-day precision
Mean amount estimated ðmgÞ in applied volume ¼ Volume of sample solution applied ðmLÞ Volume of stock solution
(2)
Amount of the drug recovered (mg) and % recovery was calculated and results of recovery studies and statistically are shown in Tables 4 and 5.
2.11.
Inter-day precision was determined by analyzing Gel sample solutions on three different days. Gel sample solution was prepared and analyzed in the similar manner as described in analysis of the gel formulation. Results of intra-day precision and inter-day precision are shown in Tables 6 and 7, respectively.
2.12. (LOQ)
Precision
2.11.1. Intra-day precision Intra-day precision was determined by analyzing Gel sample solutions at different time intervals on the same day. Gel sample solution was prepared and analyzed in the similar manner as described under analysis of the gel formulation.
Table 6 e Intra-day precision data. a
Limit of detection (LOD) and limit of quantitation
The LOD and LOQ were separately determined which is based on the standard deviation of response of the calibration curve. The standard deviation of y-intercept and slope of the calibration curves were used to calculate the LOD and LOQ. Results are shown in Table 8.
Table 7 e Inter-day precision data.
Drug
% Mean
S. D.
C. V.
Drug
% Meana
S.D.
C.V.
KETO MP PP
99.91 99.94 99.89
0.3110 0.2981 0.5912
0.3212 0.2012 0.6073
KETO MP PP
99.15 100.02 99.65
1.7061 0.4173 1.3012
1.7170 0.4153 1.3007
a Average of six determinations.
a Average of six determinations.
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2.13.
Table 8 e LOD and LOQ. Parameter
KETO
MP
PP
Limit of detection (ng/band) Limit of quantification (ng/band)
138.41 418.15
58.15 108.14
24.16 68.15
Table 9 e Result of robustness study.
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Robustness of method
To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of change in flow rate and mobile phase ratio on retention time and tailing factor were studied. The solution containing 25 mg/mL of KETO, 12.5 mg/mL of MP and 0.5 mg/mL of PP was injected (in triplicate) into sample injector of HPLC three times under the varied conditions. Robustness data is given in Table 9.
Chromatographic changes Factor Mobile phase composition (0.1 mL) 2.4:3.4:3.9 2.5:3.5:4 2.6:3.6:4.1 Amount of mobile phase (v/v) (1 mL) 9 10 11
Level
e0.1 0 þ0.1
e1.0 0 þ1.0
Rf value KETO
MP
PP
0.32 0.34 0.35
0.64 0.65 0.68
0.70 0.71 0.74
KETO
MP
PP
0.33 0.34 0.36
0.65 0.65 0.67
0.71 0.71 0.75
3.
Forced degradation study of ketoprofen
Amount of gel equivalent to about 25 mg KETO was separately transferred to five different 25.0 mL volumetric flasks (Flask no. 1, 2, 3, 4 and 5), added 5.0 mL of 0.1 M HCl, 0.1 M NaOH and 3% H2O2 to Flask no. 1, 2 and 3, respectively. Solution in flask no. 1, 2, and 3 were heated in water bath for 3 h at 80 C. Flask no. 4 containing gel was kept at 60 C for 24 h to study the effect of heat on Gel sample (heat degradation). The forced degradation was performed in the dark to exclude the possible degradative effect of light. Flask no. 5 was exposed to ultraviolet radiations at 254 nm for 24 h in a UV-chamber. All the
Fig. 9 e Chromatogram of 0.1 M NaOH gel formulation.
Fig. 10 e Chromatogram of 0.1 M HCl gel formulation.
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Fig. 11 e Chromatogram of H2O2 (3%) gel formulation.
Fig. 12 e Chromatogram of dry heat gel formulation. flasks were removed Gel samples were treated and analyzed in similar manner as described under analysis of gel formulation. The typical densitogram is shown in Figs. 9e13for acidic, alkaline, oxide, heat and UV exposure, respectively. Results of forced (stress) degradation studies are shown in Table 10.
4.
Results and discussion
In the present work, new method namely, simultaneous equation method and quick high-performance liquid
chromatography (HPLC) method were developed and validated for the simultaneous determination of three compounds in a formulated gel. The three drugs were satisfactorily resolved with Rf values of 0.33 0.05, 0.54 0.05, 0.71 0.05 for Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Calibration curves were polynomial in the range 200e1000 ng/ band, 200e1500 ng/band, 100e600 ng/band, for Ketoprofen, Methyl Paraben, and Propyl Paraben respectively. Correlation coefficient (r) values were 0.9917, 0.9927, 0.9906 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. A low relative standard deviation (<2%) was found for both precision and robustness study showing that the proposed method was precise and robust. The method had an accuracy of 99.96%, 99.91%
Fig. 13 e Chromatogram of UV radiation gel formulation.
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Table 10 e Results of degradation study. Sr. Stress condition Percent assay of no. active substance (ketoprofen) 1. 2. 3. 4.
Acid (0.1 M HCl) Alkali (0.1 M NaOH) Oxide (3% H2O2) Heat (60 C)
99.65 99.53 97.76 97.82
5.
UV (240 nm)
98.69
Acknowledgment Rf value of degraded product
0.33, 0.49, 0.67 0.33, 0.72 0.12, 0.34, 0.50, 0.64 0.34, 0.51, 0.64, 0.66, 0.78 0.33,0.58,0.77
and 101.05 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Method had the potential to determine these drugs simultaneously from dosage forms without any interference, in accordance with ICH guidelines. The limit of detection was 138.41 ng/band, 58.15 ng/band and 24.16 ng/band for KETO, MP and PP respectively and limit of quantification was 418.15 ng/band, 108.14 ng/band and 68.15 ng/band for KETO, MP and PP respectively and the method was found to be specific. The percentage recovery ranges from 99 to 101%. Forced degradation conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH guideline Q1A (R2). The drug showed instability in acid and oxide, while it remained stable in neutral conditions.
5.
Conclusion
The proposed method for simultaneous estimation (HPLC) of Ketoprofen, Methyl Paraben and Propyl Paraben in their formulated gel dosage and validated as per ICH guidelines. Moreover the method is economic, simple and rapid, hence can be employed for routine analysis in quality control laboratories.
Conflicts of interest All authors have none to declare.
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I sincerely thank Zim Laboratory, Nagpur, Maharashtra and Gen Pharmaceuticals, Pune, Maharashtra for providing me the gift sample of KETO, MP and PP and I thank my lab technicians for their contribution.
references
1. Proniuk S, Lerkpulsawad S, Blanchard J. J Chromatogr Sci. 1998;36:495e498. 2. Mannucci C, BeRfini J, Cocchini A, Perico A, Salvagnini F, Triolo A. J Liq Chromatogr. 1992;15:327e335. 3. Maurer HH, Tauvel FX, Kraemer T. J Anal Toxicol. 2001;25:237e244. 4. Hoegger D, Freitag R. J Chromatogr A. 2001;914:211e222. 5. Ye ML, Zou HF, Lei ZD, Wu RN, Liu Z, Ni JY. Electrophoresis. 2001;22:518e525. 6. Baeyens WRG, Van der Weken G, Van Overbeke A, Corveleyn S, Remon JP, Deprez P. Biomed Chromatogr. 1998;12:167e169. 7. Pietta P, Manera E, Ceva P. J Chromatogr. 1987;390:454e457. 8. Vial J, Menier I, Jardy A, Amger P, Brun A, Burbaud L. J.Chromatogr B. 1998;708:131e143. 9. Abdel-Hamid ME, Novotny L, Hamza H. J Pharm Biomed Anal. 2001;24:587e594. 10. Eichhold TH, Bailey RE, Tanguay SL, Hoke SH. J Mass Spectrom. 2000;35:504e511. 11. Molero-MonfoRf M, Escuder-GilabeRf L, VillanuevaCamanas RM, Sagrado S, Medina-Hernandez MJ. J Chromatogr B. 2001;753:225e236. 12. Solich P, Hajkova R, Pospisilova M, Sicha J. Chromatographia. 2002;55:181e184. 13. Hajkova R, Solich P, Pospisilova M, Sicha J. Anal Chim Acta. 2002;467:91e96. 14. Arancibia JA, Boldrini MA, Escandar GM. Talanta. 2000;52:261e268. 15. The European Pharmacopoeia. 4th ed. (Suppl. 4.3). Strasbourg, France: Council of Europe; 2003:3065e3066. 16. Kazymyrko VK, Ivanytska LM, Davydov IA. Ukr Rheumat J. 2000;4:34. 17. Dvora´k J, Ha´jkova´ R, Matysova´ L, Nova´kova´ L, Koupparis MA, Solicha P. J Pharm Bio Ana. 2004;36:625e629.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Stability-indicating HPTLC method for simultaneous determination of Ketoprofen, Methyl Paraben and Propyl Paraben in gel formulation Pallavi Mangesh Patil a,*, Sagar Baliram Wankhede b, Praveen Digambar Chaudhari c a
Centre for Research and Development, Prist University, Vallam, Thanjavur, Tamil Nadu 613403, India Department of Pharmaceutical Chemistry, Padm. Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune 411018, Maharashtra, India c Department of Pharmaceutics, P.E. Society’s Modern College of Pharmacy, Yamunanagar, Nigdi, Pune 411044, Maharashtra, India b
article info
abstract
Article history:
Aim: A novel and quick HPTLC-densitometric method was developed for the simultaneous
Received 20 August 2013
determination of Ketoprofen, Methyl Paraben, and Propyl Paraben.
Accepted 2 September 2013
Methods: Chromatographic separation of the drugs was performed on precoated silica gel 60
Available online 26 September 2013
F254 Merck plates using Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) as a mobile phase. A TLC scanner set at 265 nm was used of Ketoprofen, Methyl Paraben, Propyl
Keywords:
Paraben respectively were validated according to ICH guidelines. Forced degradation con-
Ketoprofen (KETO)
ditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress,
Methyl Paraben (MP)
as suggested in the ICH guideline Q1A (R2).
Propyl Paraben (PP)
Results: The three drugs were satisfactorily resolved with Rf values of 0.33 0.05,
HPTLC
0.54 0.05, 0.71 0.05 for Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Cali-
Force degradation studies
bration curves were polynomial in the range 200e1000 ng/band, 200e1500 ng/band, 100 e600 ng/band, for Ketoprofen, Methyl Paraben, and Propyl Paraben respectively. Correlation coefficient (r) values were 0.9917, 0.9927, 0.9906 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. The percentage recovery ranges from 99 to 101%. Conclusion: A low relative standard deviation (<2%) was found for both precision and robustness study showing that the proposed method was precise and robust. The method had an accuracy of 99.95%, 99.85% and 100.07 of Ketoprofen, Methyl Paraben, Propyl Paraben respectively were validated according to ICH guidelines. The drug showed instability in oxide , heat and UV light, while it remained stable in neutral conditions. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. Tel.: þ91 9823720695 (mobile). E-mail addresses: [email protected], [email protected] (P.M. Patil). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.09.004
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1.
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Introduction
A new pharmaceutical preparation (gel) containing ketoprofen (Fig .1) as an active compound with anti-inflammatory and analgesic activity was developed for treatment of diseases of the muscolo-skeletal apparatus, in which a local action is preferred. In order to prevent bacterial growth during the storage of the formulation,1,2 two commonly used preservativesda mixture of the methyl ester and propyl ester of phydroxybenzoic acid Methyl Paraben (MP) (Fig. 2) and Propyl Paraben (PP) (Fig. 3)dhave been used gas chromatographyemass spectrometry (GCeMS),3 capillary electro chromatography,4,5 high-performance liquid chromatography (HPLC) 6e8 , HPLCeMS9,10 or micellar chromatography11 as well. Only one HPLC method has been found in literature12 for simultaneous determination of KP and its degradation products, but not in the presence of preservatives. Recently, preservatives in pharmaceuticals have to be quantified. HPLC analysis of MP and PP is frequently described in the literature13e15; another publication deals with simultaneous quantification of Ketoprofen and Parabens in a commercial gel formulation by RPeHPLC with UV detection,16 but there is no any HPTLC method describing simultaneous determination of all three componentsdketoprofen, MP and PPdin pharmaceutical preparations with no any HPTLC method describing simultaneous determination in this mobile phase with beneficial system suitability parameter. For such a formulation, a novel method capable to analyze simultaneously the active component ketoprofen, and its two preservatives Methyl Paraben and Propyl Paraben was developed. Thereafter, this HPTLC method17 was successfully applied for the separation, quantification and stability study of all these compounds in formulated ketoprofen gel 2.5%.
Fig. 1 e Ketoprofen.
Fig. 2 e Methyl Paraben.
2.2.
Preparation of standard stock solutions
2.2.1.
Stock solution A
Accurately weighed quantity (100 mg) of KETO was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.2.2.
Stock solution B
2.
Material and method
Accurately weighed quantity (100 mg) of MP was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.1.
Reagents and chemicals
2.2.3.
Toluene, Ethyl acetate, Glacial acetic acid from S. D. Fine Chemicals, Mumbai Reference standard Ketoprofen and Methyl Paraben and Propyl Paraben were procured from ZIM laboratories, Nagpur, India as gift samples. Formulated gel formulation (Ketoprofen 2.5% w/w). Instrumentation and chromatographic conditions are given in the following table:
Sr. no.
Instruments
Descriptions
1 2 3 4 5
HPTLC system Sample application Scanner Software Saturated chamber
6
HPTLC plate
7
Syringe
Camag HPTLC system Camag Linomat IV automatic sample Camag TLC scanner Camag winCATS software Camag twin-trough chamber (10 10) and (20 20) Merck HPTLC plate coated with silica gel 60 F 254 (0.2 mm thickness) on aluminum sheet Hamilton syringe (100 ml)
Stock solution C
Accurately weighed quantity (100 mg) of PP was transferred to 100.0 mL volumetric flask, dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 50.0 mL volumetric flask and diluted to the mark with mobile phase (concentration 100 mg/mL). The solution was mixed and filtered through 0.2 m membrane filter.
2.2.4.
Stock solution D
An accurately weighed quantity of 250 mg KETO and 100 mg MP, 10 mg was transferred to 100.0 mL volumetric flasks, 40.0 mL of mobile phase was added; the content was dissolved and diluted up to the mark with mobile phase. From this solution, 5.0 mL was transferred to 10.0 mL volumetric flask and diluted to the mark with mobile phase. Further, 5.0 mL of
Fig. 3 e Propyl Paraben.
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above solution was diluted to 10.0 mL with mobile phase (concentration of 625 mg/mL KETO and 250 mg/mL MP, 25 mg/mL PP respectively). The solution was mixed and filtered through 0.2 m membrane filter.
2.3.
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Migration distance: z80 mm Temperature: 20 5 C Scanning mode: Absorbance/Reflectance Slit dimensions: 5 0.45 mm Scanning wavelength: 265 nm
Selection of mobile phase The retention factors of KETO, MP and PP were:
Aliquot portion of standard stock solutions D (5 mL each) was applied on TLC plates in the form of band (band size: 6 mm). Different solvents with varying polarity as well as combination of solvent were tried to get well separated bands of the drugs. After trying several permutations and combinations, the solvent system containing Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) was found to be most satisfactory as it gave good resolution of both drugs.
2.4. Selection of wavelength for densitometric evaluation of separated bands Standard stock solution D was applied on TLC plate with the help of CAMAG LINOMAT-V automatic sample applicator, the plate was chromatographed in twin-through glass chamber saturated with mobile phase for 30 min. After chromatographic development, the plate was removed and air dried. The separated bands on the TLC plate were scanned over the wavelength range of 200e700 nm. The wavelength 265 nm was selected for densitometric evaluation of separated bands. The overlain spectrum obtained is depicted in Fig. 4.
2.5.
Optimum chromatographic conditions
Stationary phase: Aluminum plates precoated with silica gel 60 F254 (Merck) Mobile phase: Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) Plate size: 10 cm 10 cm Mode of application: Band Band size: 6 mm (Distance between two bands: 5.5 mm) Sample volume: 5 mL Development chamber: Twin-through glass chamber, 10 cm 10 cm with (20 20) stain less steel lid Saturation time: 30 min Se´paration technique: Ascending
KETO: 0.33 0.05 MP: 0.54 0.05 PP: 0.71 0.05 Densitogram of KETO, MP and PP is shown in Fig. 5.
2.6.
Study of linearity range
The standard stock solution A containing KETO and standard stock solution B containing MP and stock solution C containing PP was applied on the TLC plate in the range 1e6 mL with the help of micro syringe using LINOMAT-V automatic sample applicator. The plate was then developed and scanned under the above mentioned chromatographic conditions. Rf was recorded for each drug concentration and the calibration curves of the concentration vs. Rf were constructed for both the drugs. The calibration curve for KETO and MP and PP are depicted in Figs. 6e8 respectively.
2.6.1.
Selection of mobile phase
From standard stock D was appropriately to obtain final concentration 625 mg/mL KETO and 250 mg/mL MP, 25 mg/mL PP respectively. The diluted standard solutions were filtered through 0.2 m membrane filter. After trying several permutations and combinations, the solvent system containing Toluene:Ethyl acetate:Glacial acetic acid (6.5:2.5:1.0 v/v/v) was found to be most satisfactory as it gave good resolution of both drugs.
2.7.
Preparation of gel formulation of Ketoprofen
Ketoprofen gel formulation was prepared using 1% carbopol 940 and as a gelling agent. Gelling agent was dispersed in a small quantity of distilled water 75 ml and then stored overnight to ensure complete hydration. Ketoprofen in a suitable
Fig. 4 e Overlain spectra of KETO, MP and PP.
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Fig. 5 e Typical densitogram of KETO, MP and PP.
solvent (water) as added to the dispersion and make up weight with distilled water. Other excipients (Methyl Paraben 1% and propyl Paraben 0.1%) were also added slowly with continuous stirring. In carbopol gels, pH of the vehicle was brought to neutral by using TEA (Triethanolamine). The final weight of the gel was adjusted to 100 gm with distilled water. Entrapped air bubbles were removed by keeping the gels in vacuum desiccators as shown in Table 1.
2.8.
Analysis of gel formulation
An accurately weighed quantity of gel was weighed equivalent to about 1000 mg of Ketoprofen and 400 mg of Methyl Paraben and 40 mg Propyl Paraben into a 1000 mL volumetric flask. And appropriate amount of methanol was then added. The mixture was ultrasonicated for 30 min with heating and allowed to cool at room temperature before adjusting to volume with methanol. The organic layer was decanted and the extraction procedure was repeated. The resulting mixture was centrifuged at 3500 rpm for 20 min and 20 mL of the clear supernatant was injected directly
Fig. 6 e Standard calibration curve for Ketoprofen.
onto the column. After centrifugation 20 mL of this mixture was injected into the chromatograph. The resulting solution was mixed and filtered through Whatman filter paper and filtrate was appropriately diluted to get approximate concentration and to obtain final concentration of 1000 mg/mL KETO and 400 mg/mL MP, 40 mg/mL respectively. The diluted solution was filtered through 0.20 m filter. On the TLC plate two bands of standard stock solution D and four bands of sample solution, 5.0 mL each, were applied and the plate was developed and scanned under the optimum chromatographic condition. After chromatographic development the peak obtained for standard and sample bands was integrated. The amount of KETO, MP and PP present in applied volume of standard solution was fed to computer. Amount of drug present in applied volume of sample solution was obtained by comparing Rf of sample bands with that of standard bands. Amount of drug estimated in mg/gel and the percent label claim were calculated using the following formula: The content of KETO, MP and PP in sample was calculated using the following formula no. 1.
Fig. 7 e Standard calibration curve for Methyl Paraben.
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2.9.1.
Accuracy
To as certain the accuracy of proposed method, recovery studies were carried out by standard addition method, as per ICH guidelines.
2.10.
Preparation of sample solutions
An accurately weighed quantity of pre-analyzed gel equivalent to about 1000 mg KETO, 400 mg MP and 40 mg PP was transferred individually in nine different 1000.0 mL volumetric flasks. To each of the flask following quantities of KETO, MP and PP were added: Fig. 8 e Standard calibration curve for Propyl Paraben.
Table 1 e Composition of the carbopol and pure drug Ketoprofen as below. Ingredient
Quantity taken
Ketoprofen Methyl Paraben Propyl Paraben Carbopol (1%) as gel base Double distilled water Triethanolamine
2.5 g 1.0 g 0.1 g QS Make up to 100 ml Q S to neutralize gel
Q.S (Quality sufficient).
Amount of drug estimated ðmg=gelÞ ¼
Mean amount estimated ðmgÞ in applied volume Volume of sample solution applied ðmLÞ
Volume of stock solution ðmLÞ Wt: of gel taken ðmgÞ
Average wt: of gel ðmgÞ
(1)
Percent label claim was calculated using above formula no 1. Results of analysis of gel formulation and its statistical evaluation are shown in Tables 2 and 3 respectively.
2.9.
Method validation
The proposed method was validated by studying several parameters such as accuracy, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and robustness.
Flask no.1: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.2: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.3: 800 mg KETO þ 320 mg MP þ 32 mg PP Flask no.4: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.5: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.6: 1000 mg KETO þ 400 mg MP þ 40 mg PP Flask no.7: 1200 mg KETO þ 480 mg MP þ 48 mg PP Flask no.8: 1200 mg KETO þ 480 mg MP þ 48 mg PP Flask no.9: 1200 mg KETO þ 480 mg MP þ 48 mg PP Then 100 mL methanol was added to each flask and content of the flask was ultrasonicated for 20 min, volume was then made up to the mark with mobile phase. The solution was individually mixed and filtered through Whatman filter paper no. 42. From the filtrate, 1.0 mL solution was diluted to 10.0 mL with mobile phase. The diluted solution was filtered through 0.2 m membrane filter. On the TLC plate two bands of standard stock solution D and four bands of sample solution, 5.0 mL each, were applied and the plate was developed and scanned under the optimum chromatographic condition. After chromatographic development the peak obtained for standard and sample bands were integrated. The amount of KETO, MP and PP present in applied volume of standard solution was fed to computer. Amount of drug present in applied volume of sample solution was obtained by comparing Rf of sample bands with that of standard bands. Amount of KETO, MP and PP in sample was calculated by comparing the mean Rf for standard and sample solution by formula no. 2. Amount of KETO, MP and PP in sample (mg) was calculated by following formula:
Table 2 e Results of analysis gel formulation. Formulated gel Sr. no.
1. 2. 3. 4. 5. 6.
Average weight: 4.000 mg
Weight of gel taken (mg)
4000 4000 4000 4000 4000 4000
Amount of drug estimated (mg/Gel taken)
% Label claim
KETO
MP
PP
KETO
MP
PP
1000.09 1000.01 1000.00 999.98 999.78 1000.01
399.00 399.12 400.00 400.12 398.78 401.02
40.00 39.87 39.87 40.00 41.02 41.77
100.90 100.49 100.00 99.88 99.79 100.03
99.80 99.87 100.00 100.12 99.88 100.73
100.00 99.89 99.89 100.00 101.00 101.01
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Table 3 e Statistical validation for analysis of gel formulation. Sr. no.
Drug
Amount of drug estimated (mg/gel)a
% Label claima
S.D
C.V
S.E
1. 2. 3
KETO MP PP
999.99 399.67 40.42
99.95 99.85 100.00
1.3143 1.2108 0.2147
1.4278 1.2146 0.2251
0.3181 0.1504 0.1151
a Mean of six determinations.
Table 4 e Results of recovery studies. Level of recover
Weight of gel taken (mg)
80%
Amount of drug added (mg)
Amount of drug recovered (mg)
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
800 800 800 1000 1000 1000 1200 1200 1200
320 320 320 400 400 400 480 480 480
32 32 32 40 40 40 48 48 48
799.78 800.01 800.12 999.18 999.70 1000.10 1198.78 1200.00 1201.03
318.13 319.79 320.11 399.18 399.77 400.18 479.11 480.72 480.75
31.99 32.00 32.11 40.10 39.98 40.11 47.91 47.95 48.01
99.71 100.01 100.19 99.78 99.88 100.02 99.93 100.00 101.02
98.11 99.89 100.11 99.77 99.85 100.11 99.10 100.42 100.47
99.98 100.00 100.12 100.11 99.92 100.12 99.92 99.95 100.01
4000 4000 4000 4000 4000 4000 4000 4000 4000
100%
120%
% Recovery
Table 5 e Statistical validation for recovery study. % Mean recoverya
Level of recovery
80% 100% 120%
Standard deviation
% R.S.D.
S.E
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
KETO
MP
PP
99.97 99.95 100.02
99.89 99.85 100.15
100.10 100.05 100.15
1.2679 1.2454 1.2788
1.2488 1.2214 1.2958
1.3011 1.3117 1.2481
1.2519 1.2316 1.2668
1.2310 1.2161 1.2848
1.2923 1.3109 1.2316
0.6111 0.5922 0.7155
0.5977 0.5811 0.7066
0.7122 0.7211 0.6811
a Mean of three determinations.
Amount of drug KETO; MP and PP ðmLÞ estimated ðmgÞ
2.11.2. Inter-day precision
Mean amount estimated ðmgÞ in applied volume ¼ Volume of sample solution applied ðmLÞ Volume of stock solution
(2)
Amount of the drug recovered (mg) and % recovery was calculated and results of recovery studies and statistically are shown in Tables 4 and 5.
2.11.
Inter-day precision was determined by analyzing Gel sample solutions on three different days. Gel sample solution was prepared and analyzed in the similar manner as described in analysis of the gel formulation. Results of intra-day precision and inter-day precision are shown in Tables 6 and 7, respectively.
2.12. (LOQ)
Precision
2.11.1. Intra-day precision Intra-day precision was determined by analyzing Gel sample solutions at different time intervals on the same day. Gel sample solution was prepared and analyzed in the similar manner as described under analysis of the gel formulation.
Table 6 e Intra-day precision data. a
Limit of detection (LOD) and limit of quantitation
The LOD and LOQ were separately determined which is based on the standard deviation of response of the calibration curve. The standard deviation of y-intercept and slope of the calibration curves were used to calculate the LOD and LOQ. Results are shown in Table 8.
Table 7 e Inter-day precision data.
Drug
% Mean
S. D.
C. V.
Drug
% Meana
S.D.
C.V.
KETO MP PP
99.91 99.94 99.89
0.3110 0.2981 0.5912
0.3212 0.2012 0.6073
KETO MP PP
99.15 100.02 99.65
1.7061 0.4173 1.3012
1.7170 0.4153 1.3007
a Average of six determinations.
a Average of six determinations.
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2.13.
Table 8 e LOD and LOQ. Parameter
KETO
MP
PP
Limit of detection (ng/band) Limit of quantification (ng/band)
138.41 418.15
58.15 108.14
24.16 68.15
Table 9 e Result of robustness study.
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Robustness of method
To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of change in flow rate and mobile phase ratio on retention time and tailing factor were studied. The solution containing 25 mg/mL of KETO, 12.5 mg/mL of MP and 0.5 mg/mL of PP was injected (in triplicate) into sample injector of HPLC three times under the varied conditions. Robustness data is given in Table 9.
Chromatographic changes Factor Mobile phase composition (0.1 mL) 2.4:3.4:3.9 2.5:3.5:4 2.6:3.6:4.1 Amount of mobile phase (v/v) (1 mL) 9 10 11
Level
e0.1 0 þ0.1
e1.0 0 þ1.0
Rf value KETO
MP
PP
0.32 0.34 0.35
0.64 0.65 0.68
0.70 0.71 0.74
KETO
MP
PP
0.33 0.34 0.36
0.65 0.65 0.67
0.71 0.71 0.75
3.
Forced degradation study of ketoprofen
Amount of gel equivalent to about 25 mg KETO was separately transferred to five different 25.0 mL volumetric flasks (Flask no. 1, 2, 3, 4 and 5), added 5.0 mL of 0.1 M HCl, 0.1 M NaOH and 3% H2O2 to Flask no. 1, 2 and 3, respectively. Solution in flask no. 1, 2, and 3 were heated in water bath for 3 h at 80 C. Flask no. 4 containing gel was kept at 60 C for 24 h to study the effect of heat on Gel sample (heat degradation). The forced degradation was performed in the dark to exclude the possible degradative effect of light. Flask no. 5 was exposed to ultraviolet radiations at 254 nm for 24 h in a UV-chamber. All the
Fig. 9 e Chromatogram of 0.1 M NaOH gel formulation.
Fig. 10 e Chromatogram of 0.1 M HCl gel formulation.
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Fig. 11 e Chromatogram of H2O2 (3%) gel formulation.
Fig. 12 e Chromatogram of dry heat gel formulation. flasks were removed Gel samples were treated and analyzed in similar manner as described under analysis of gel formulation. The typical densitogram is shown in Figs. 9e13for acidic, alkaline, oxide, heat and UV exposure, respectively. Results of forced (stress) degradation studies are shown in Table 10.
4.
Results and discussion
In the present work, new method namely, simultaneous equation method and quick high-performance liquid
chromatography (HPLC) method were developed and validated for the simultaneous determination of three compounds in a formulated gel. The three drugs were satisfactorily resolved with Rf values of 0.33 0.05, 0.54 0.05, 0.71 0.05 for Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Calibration curves were polynomial in the range 200e1000 ng/ band, 200e1500 ng/band, 100e600 ng/band, for Ketoprofen, Methyl Paraben, and Propyl Paraben respectively. Correlation coefficient (r) values were 0.9917, 0.9927, 0.9906 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. A low relative standard deviation (<2%) was found for both precision and robustness study showing that the proposed method was precise and robust. The method had an accuracy of 99.96%, 99.91%
Fig. 13 e Chromatogram of UV radiation gel formulation.
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Table 10 e Results of degradation study. Sr. Stress condition Percent assay of no. active substance (ketoprofen) 1. 2. 3. 4.
Acid (0.1 M HCl) Alkali (0.1 M NaOH) Oxide (3% H2O2) Heat (60 C)
99.65 99.53 97.76 97.82
5.
UV (240 nm)
98.69
Acknowledgment Rf value of degraded product
0.33, 0.49, 0.67 0.33, 0.72 0.12, 0.34, 0.50, 0.64 0.34, 0.51, 0.64, 0.66, 0.78 0.33,0.58,0.77
and 101.05 Ketoprofen, Methyl Paraben, Propyl Paraben respectively. Method had the potential to determine these drugs simultaneously from dosage forms without any interference, in accordance with ICH guidelines. The limit of detection was 138.41 ng/band, 58.15 ng/band and 24.16 ng/band for KETO, MP and PP respectively and limit of quantification was 418.15 ng/band, 108.14 ng/band and 68.15 ng/band for KETO, MP and PP respectively and the method was found to be specific. The percentage recovery ranges from 99 to 101%. Forced degradation conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH guideline Q1A (R2). The drug showed instability in acid and oxide, while it remained stable in neutral conditions.
5.
Conclusion
The proposed method for simultaneous estimation (HPLC) of Ketoprofen, Methyl Paraben and Propyl Paraben in their formulated gel dosage and validated as per ICH guidelines. Moreover the method is economic, simple and rapid, hence can be employed for routine analysis in quality control laboratories.
Conflicts of interest All authors have none to declare.
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I sincerely thank Zim Laboratory, Nagpur, Maharashtra and Gen Pharmaceuticals, Pune, Maharashtra for providing me the gift sample of KETO, MP and PP and I thank my lab technicians for their contribution.
references
1. Proniuk S, Lerkpulsawad S, Blanchard J. J Chromatogr Sci. 1998;36:495e498. 2. Mannucci C, BeRfini J, Cocchini A, Perico A, Salvagnini F, Triolo A. J Liq Chromatogr. 1992;15:327e335. 3. Maurer HH, Tauvel FX, Kraemer T. J Anal Toxicol. 2001;25:237e244. 4. Hoegger D, Freitag R. J Chromatogr A. 2001;914:211e222. 5. Ye ML, Zou HF, Lei ZD, Wu RN, Liu Z, Ni JY. Electrophoresis. 2001;22:518e525. 6. Baeyens WRG, Van der Weken G, Van Overbeke A, Corveleyn S, Remon JP, Deprez P. Biomed Chromatogr. 1998;12:167e169. 7. Pietta P, Manera E, Ceva P. J Chromatogr. 1987;390:454e457. 8. Vial J, Menier I, Jardy A, Amger P, Brun A, Burbaud L. J.Chromatogr B. 1998;708:131e143. 9. Abdel-Hamid ME, Novotny L, Hamza H. J Pharm Biomed Anal. 2001;24:587e594. 10. Eichhold TH, Bailey RE, Tanguay SL, Hoke SH. J Mass Spectrom. 2000;35:504e511. 11. Molero-MonfoRf M, Escuder-GilabeRf L, VillanuevaCamanas RM, Sagrado S, Medina-Hernandez MJ. J Chromatogr B. 2001;753:225e236. 12. Solich P, Hajkova R, Pospisilova M, Sicha J. Chromatographia. 2002;55:181e184. 13. Hajkova R, Solich P, Pospisilova M, Sicha J. Anal Chim Acta. 2002;467:91e96. 14. Arancibia JA, Boldrini MA, Escandar GM. Talanta. 2000;52:261e268. 15. The European Pharmacopoeia. 4th ed. (Suppl. 4.3). Strasbourg, France: Council of Europe; 2003:3065e3066. 16. Kazymyrko VK, Ivanytska LM, Davydov IA. Ukr Rheumat J. 2000;4:34. 17. Dvora´k J, Ha´jkova´ R, Matysova´ L, Nova´kova´ L, Koupparis MA, Solicha P. J Pharm Bio Ana. 2004;36:625e629.